No abstract

The development of genetically encoded fluorescent voltage probes is essential to image electrical activity from neuronal populations. Previous green fluorescent protein (GFP)-based probes have had limited success in recording electrical activity of neurons because of their low sensitivity and poor temporal resolution. Here we describe a hybrid approach that combines a genetically encoded fluorescent probe (membrane-anchored enhanced GFP) with dipicrylamine, a synthetic voltage-sensing molecule that partitions into the plasma membrane. The movement of the synthetic voltage sensor is translated via fluorescence resonance energy transfer (FRET) into a large fluorescence signal (up to 34% change per 100 mV) with a fast response and recovery time (0.5 ms). Using this two-component approach, we were able to optically record action potentials from neuronal cell lines and trains of action potentials from primary cultured neurons. This hybrid approach may form the basis for a new generation of protein-based voltage probes.

Synapsins, a family of synaptic vesicle proteins, have been shown to regulate neurotransmitter release; the mechanism(s) by which they act are not fully understood. Here we have studied the role of domain E of synapsins in neurotransmitter release at the squid giant synapse. Two squid synapsin isoforms were cloned and found to contain a carboxy (C)-terminal domain homologous to domain E of the vertebrate a-type synapsin isoforms. Presynaptic injection of a peptide fragment of domain E greatly reduced the number of synaptic vesicles in the periphery of the active zone, and increased the rate and extent of synaptic depression, suggesting that domain E is essential for synapsins to regulate a reserve pool of synaptic vesicles. Domain E peptide had no effect on the number of docked synaptic vesicles, yet reversibly inhibited and slowed the kinetics of neurotransmitter release, indicating a second role for synapsins that is more intimately associated with the release process itself. Thus, synapsin domain E is involved in at least two distinct reactions that are crucial for exocytosis in presynaptic terminals.